Multiple sclerosis (MS) is a multifocal demyelinating disease that results in neural dysfunction and culminates in physical and cognitive disability. During MS, immune cells attacks the myelin sheath causing inflammation, axonal damage, and impaired remyelination. Repair is inefficient in the presence of misfolded proteins, protein aggregates, damaged organelles, and myelin debris, resulting in chronic disability and neurodegeneration. Therefore, it is important to define the damaged cellular components and understand how to efficiently eliminate them from CNS lesions that develop during MS. Protein aggregates are highly resistant to protein degradation and targeting them for clearance is challenging. The long-term goal is to identify the proteins within aggregates and understand how to eliminate them for efficient clearance of debris and better repair. Cells attempt to remove misfolded proteins by degrading, refolding, or sequestering them in intracellular compartments. If the proteins and the mechanisms by which they are degraded are identified, the efficiency of debris clearance can be improved with targeted therapy. In this proposal, we will test the hypothesis that following sensitization with MOG35-55 neuronal/axonal proteins and proteins enriched in the myelin sheath are inefficiently cleared and form aggregates. The rationale for this project is the observation that protein of unknown composition form aggregates in MS lesions. The central hypothesis will be tested by pursuing two specific aims. Specific Aim 1 will characterize and validate the proteins within aggregates during acute and chronic EAE. As part of our study, we will compare protein aggregates from age- and sex-matched mouse spinal cord and identify the proteins by mass spectrometry (nanoLC-mass spectrometry ms/ms). This analysis will identify common functions and conserved motifs within the identified proteins and compare functions and clearance pathways using bioinformatics analyses, and western blot analysis. Specific Aim 2 will examine how clearance by the proteasome and macroautophagy (autophagy) affects protein aggregate accumulation. Treatment with a disaccharide shown to activate autophagy will determine whether treatment is able to reduce the number of proteins associated with aggregates as well as reduce clinical scores of treated mice. The research proposed is innovative because it provides a framework to identify and assess proteins and the signaling pathways associated with insoluble aggregates observed in EAE. The proposed research is significant because it is expected to provide scientific justification to address how to clear protein aggregates, improve repair, and identify target areas for therapy designed to efficiently clear cellular clearance and reduce the duration in which axons are exposed to an unfavorable milieu in the CNS.